1. Field of the Invention
The present invention relates to a method for selectively amplifying a cDNA. More specifically, the invention is concerned with a method for selectively amplifying a cDNA synthesized from an mRNA expressed only in an extremely small quantity.
2. Prior Art
Inherited diseases, cancers and some types of progeria and dementia are caused by occurrence of variation in specific genes. Thus, these diseases are often called generically "genetic diseases". In these genetic diseases, abnormal characters appear only in such an organ or tissue where a specific gene involved in the disease is expressed. However, the variation in the gene itself is present in any tissue in the body. Therefore, as a technique for diagnosing a genetic disease, a method may be considered which comprises extracting genomic DNA from leukocytes in peripheral blood that is most easy to sample and then amplifying the genomic DNA.
As a method for amplifying genomic DNA, firstly, a method by PCR (polymerase chain reaction) may be given. Then, a genetic disease is diagnosed by searching the amplified DNA for variation in the base sequence.
However, it is difficult in general to amplify an entire genomic DNA by performing PCR just one time because the size of genomic DNA is too big.
As a means to amplify genomic DNA, secondly, a method of amplifying exons alone may be given because variation in bases involved in a genetic disease only occurs in the region of "exons" encoding proteins. In order to amplify exons alone, information on genetic structure, i.e. information as to which regions of the genomic DNA are exons is necessary.
However, it is seldom that such information has been previously known.
Due to these reasons, in many cases it is necessary to use as a sample for genetic diagnosis not peripheral blood but a sample obtained by biopsy from a tissue manifesting symptoms. Generally, mRNA (precursors of proteins) is extracted from the tissue sample obtained and cDNA is synthesized therefrom. Subsequently, a PCR is performed (this method is called "RT-PCR").
However, biopsy gives a great pain to a patient and, moreover, some tissues such as brain are extremely difficult to perform biopsy.
For the reasons described above, it is the present situation that genetic diagnosis using easy-to-sample blood has many restrictions, and that many restrictions are also present in the case of examining a tissue obtained by biopsy to find out at which part of the coding region of a candidate causative gene of an inherited disease or the like variation in specific base sequences has occurred.
On the other hand, recently, it has been made clear by RT-PCR or the like that such a gene that is expressed only tissue-specifically (e.g., in brain or heart) is actually expressed in an extremely small quantity in leukocytes in peripheral blood, fibroblasts in skin and the like (Chelly, J. et al., Proc. Natl. Acad. Sci. USA 86, 2617-2621 (1989)). It is appropriate to interpret this phrase "expressed in an extremely small quantity" that inhibition of gene expression in the body is not 100% complete but there is some "leakage", rather than to interpret this as physiological gene expression. In other words, the "expressed in an extremely small quantity" does not mean that a specific gene is expressed naturally and properly though in an extremely small quantity. It is appropriate to interpret that an mRNA being expressed in a specific tissue such as brain may leak out of the tissue though it should be expressed only in that specific tissue, and that since the body cannot inhibit the leakage of such mRNA completely, the mRNA which has leaked out in an extremely small quantity exists in tissues other than the specific tissue as well (e.g., peripheral blood leukocyte).
Such RNA which has thus leaked out is called "leaky RNA" or "illegitimate transcription". If a target cDNA can be amplified selectively from such "leaky" mRNA by RT-PCR, it is believed that, in principle, diagnosis of any genetic disease can become possible by examining peripheral blood.
In conventional RT-PCR, total RNA is extracted from a tissue and then mRNAs are isolated and purified. Thereafter, using oligo(dT) primers or random primers, a pool of cDNAs from mRNAs being expressed in the tissue is prepared (Michael A. Innis et al., PCR Protocols: A Guide to Methods and Applications, Academic Press Inc. (1990)). Then, a PCR is performed using an appropriate set of oligo DNA primers specific to a target cDNA.
Since the template for the above PCR is a mixture of cDNAs from the entire tissue, contamination with (mixing of) a large number of non-specific amplified products may occur when the amount of cDNAs or the number of PCR cycles is increased. Accordingly, it has been difficult to apply this method to the "illegitimate transcription" described above.